Project Summary A key aim of this proposal is to equip the candidate, Dr. Jamie Justice, with the expertise to become an independent investigator who can advance interventions that extend healthy lifespan to randomized, controlled trials in older persons. Specifically, cellular senescence is a biologic hallmark of aging that emerging preclinical evidence indicates could have profound consequences on aging-related disease and function, and removal of senescent cells results in robust improvements in healthspan in rodents. Translation of these interventions to clinical trial has been proposed, yet health consequences of cell senescence and therapeutic potential has not been evaluated in humans. Dr. Justice's preliminary data in a small number of older women are the first to show that cells expressing tumor suppressor protein and senescence biomarker p16INK4a are present in adipose tissue from older adults and related to worse physical function, but exercise and weight loss by caloric restriction may mitigate this burden. The proposed research project represents a critical next step by examining the effects of caloric restriction (CR) on cell senescence in a prospective randomized controlled trial (RCT). The primary hypothesis is that a CR intervention will reduce senescent cell burden and this reduction will be related to improvement in functional and metabolic outcomes. This will be accomplished by capitalizing on a recent NIH-funded RCT (VEGGIE, R01DK103531) and the candidate's engaged inter-disciplinary primary mentoring team (Drs. Nicklas, Ding, Kritchevsky, Kirkland). VEGGIE will determine the effects of CR designed to achieve 10% weight loss vs. health education control in 200 men and women aged 40-65 years with obesity (BMI 30-45 kg/m2), to characterize epigenetic and transcriptomic effects of CR in adipocytes and peripheral blood monocytes and T cells, and associations with physical and metabolic function. We propose an ancillary investigation in a subset of 90 participants (50-65 years, n=45 per grp) to determine the effects of CR on senescent cell burden (Aim 1): a) proportion of p16INK4a expressing senescent cells (immunohistochemistry) in subcutaneous abdominal adipose tissue; b) expression of senescence biomarkers in isolated adipocytes and monocytes (RNAseq) and T cells (p16INK4a expression); and c) SASP biomarkers in plasma (cytokine/chemokine panel). We will also examine cross-sectional associations of age and obesity with cell senescence (Aim 2), and relationships between changes in senescence biomarkers and physical function and metabolic outcomes (Aim 3). The research proposed is aligned with an approved NIA concept to develop markers of aging-related biologic mechanisms for human studies. Additionally, it will provide essential training for the candidate, who will establish expertise in cell senescence and translational research, and develop competencies in leading clinical trials with biological outcomes. This approach provides the ideal platform to advance the candidate's career as an independent investigator, and provide the foundation to establish the role of cell senescence in human age-related functional decline.